Project Summary Cellular metabolic energy is stored in the form of neutral lipids, particularly triacylglycerols (TGs), which are packaged in cytoplasmic lipid droplets (LDs). Excessive accumulation of LDs occurs during the progression of many diseases, including obesity, type 2 diabetes, atherosclerosis and metabolic syndrome. In addition, LDs play a key role in some infections, including hepatitis C virus and chlamydia, by facilitating replication of some pathogens. Despite their important role in human health and disease, surprisingly little is known about LDs. LDs consist of a hydrophobic core surrounded by a phospholipid monolayer. Most functions of LDs, including TG synthesis, TG storage, and energy mobilization, are executed and regulated by these surface proteins. How these proteins are specifically targeted to LDs, however, remains unclear. Among organelles, LDs are unique because their surface is at the interface of a hydrophobic phase (the LD core) and an aqueous phase (the cytoplasm) and thus are unable to accommodate typical transmembrane proteins with globular domains. Therefore, the targeting of proteins to LDs must involve unique mechanisms. In this project, we will determine how proteins are specifically targeted to the surface of LDs. We will investigate how triglyceride enzymes including GPAT4 and others re-localize to LDs, enabling their growth. In addition, we will determine how amphipathic helix-containing proteins, including the rate-limiting phosphatidylcholine enzyme CTP:phosphocholine cytidylyltransferase (CCT), are specifically targeted to the surface of LDs from the cytosol to facilitate expansion. For each part of the project, we will use cutting-edge biophysics, biochemistry, cell biology, proteomics and computational techniques and validate our findings in different model systems such as Drosophila and mammalian cells. Although the research proposed here is basic, the determination of the fundamental cellular mechanisms that drives lipid droplet protein targeting will nevertheless facilitate the development of therapeutic strategies to combat obesity, metabolic diseases and viral infections, as well as propel further research into the cell biology of these organelles.